Flavobacterium psychrophilum is a psychrotrophic fish-pathogenic bacterium that causes cold water disease (CWD) in salmonids. By means of Tn4351 mutagenesis a mutant named FP1033, deficient in growth on iron-depleted medium, was previously isolated. FP1033 recovered the parental phenotype in the presence of iron. The gene disrupted by the transposon in this mutant encoded a protein with similarity to ExbD proteins, which are members of the TonB complex system involved in iron uptake mediated by siderophores. Analysis of the DNA surrounding the transposon insertion showed the presence of a tonB cluster of genes composed of exbB, two exbD (exbD1 and exbD2) and tonB loci. RT-PCR analysis and complementation studies indicated that these genes are transcribed as an operon and that the exbD2 : : Tn4351 phenotype was caused by the lack of ExbD2. FP1033 showed decreased virulence and conferred a high level of protection in rainbow trout fry after vaccination. This is believed to be the first report of a F. psychrophilum attenuated strain that induces a protective immune response in rainbow trout against CWD. These results suggest that the exbD2 locus from this particular TonB system is a suitable target to generate a live attenuated vaccine.